ABSTRACT The experiment is conducted to study the growth kinetics of microorganism in shake flask experiment. E-coli is grown in a Terrific broth medium and being fermented for 24 hours at 350 rpm and 37°C. The cell culture is taken out every 1 hour from 0 hour to 4th hour and continued with every 2 hours until 20th hour. The absorbance analysis, glucose analysis and cell dry weight are also being performed during the experiment. For the optical density, the absorbance reading from the spectrophotometer is taken while for the glucose test, the absorbance reading of glucose level is taken from the YSI 2700 Select Biochemical Analyzer. Then, these substrate concentration values will be plotted on graph with growth rate. In the other hand, the cell dry weight is taken after the mass concentration is being dried overnight in the oven. The weight of the tube that contains the biomass before and after the drying process is recorded in order to get the cell dry weight. As for the optical density of the cell, the absorbance value showed an increment which indicating that the cell was growing and number of cell was increased in the shake flask. However, the growth rate cannot be determined as the absorbance values were increased and decreased unevenly and calculation of rate of microbial growth cannot be made as the data for cell dry weight concentration, X are not consistent. Supposedly, the cell dry weight should be increased as the number of cell increased inside the shake flask.
2.0
INTRODUCTION
Growth kinetics such as the relationship between specific growth rate and the concentration of a substrate is one of the basic tools in microbiology. Conventional growth kinetics derived from single-substrate-controlled laboratory experiments have invariably been used for describing both growth and substrate utilization in ecosystems. However, in nature, microbial cells are exposed to a wide spectrum of potential substrates, many of which they utilize simultaneously. The fermentation culture requirement includes a good culture media where the nutrient requirement is optimized to enhance the both the growth and production of end/by-products (Stephanie C. et al., 2014). Next, the temperature must be under 20°C for the normal bacteria as if it is above the optimal temperature, growth slows down and thermal death may occur. Then, the pH is another important requirement for good growth of microbial which is acceptable under pH 3-8. The dissolved oxygen is another essential substrate for aerobic fermentation. As stated by Stephanie C. et al. (2014), in this experiment, batch fermentation takes place as the fermentation is performed after sterilization of the medium and adjustment of pH by either acid or alkali. After inoculation of yeast or bacteria, product formation takes place by controlling temperature, pH, agitation, and dissolved oxygen, which depend on the characteristics of the cultured microorganism. Because no medium addition occurs, the growth of microorganism follows four main phases of the growth curve; lag phase, log (exponential phase, stationary phase, and death phase. In studying the growth kinetics of the microorganism, shake flask fermentation is one of the widely used fermentation method for screening of high producing strains. The shake flask fermentation is the simplest way to do the fermentation using small volume of nutrient broth in laboratory. A nutritionally rich medium, ‘Terrific’ broth or ‘Luria Bertani’ (LB) is used for the growth of bacteria. Normally, the shake fermentation flask used is between 250ml to 500ml range. An Erlenmeyer flask used is equipped with cotton wool stopper and autoclaved containing the chosen nutrient broth inside the flask which is “Terrific” broth.
Then, the flask is allowed to cool to the room temperature before some microbes are allowed to grown inside the flask and put into the shaker machine. Plugs made from cotton-wool, is used to prevent airborne microorganism from getting to the medium while at the same allowing free flow of air into the flask. Shaker machine is designed to assist with oxygen transfer for aerobic respiration microorganism. For more precisely defined environment, incubator shaking cabinet can be very well use. These cabinets can control the temperature, illumination, gaseous levels and humidity. The movement of shaker normally rotary shaking action or reciprocating shaking action is produced. Besides, by increasing the speed of a shaker can increase the oxygen transfer rate of a particular flask.
3.0
OBJECTIVE
The experiment is conducted to 1. To study the growth kinetics if microorganism in shake flask experiment. 2. To construct a growth curve including lag, log, stationary and death phases. 3. To determine the Monod parameters of maximum growth rate (μ max), yield of substrate (Yx/s), mass doubling time (t d), saturation constant (Ks), specific rate (μnet).
4.0
THEORY
Genetically modified Escherichia coli have been chosen as the host organism for each of the co-proteins to be produced (Shuler & Kargi, 2002). Each strain of E. coli will contain a different gene that is responsible for producing the desired co-protein. The modified E. coli cells will be separately cells grown through the process of batch fermentation. A microbial culture is a method of multiplying microbial organisms by letting reproduce in predetermined culture media under controlled laboratory condition (Benjamin C., 2004). The relation between the specific growth rate (µ) of a population of microorganisms and the substrate concentration (S) is a valuable tool in biotechnology. This relationship is represented by a set of empirically derived rate laws referred to as theoretical models to describe the behaviour of a given system.
The generation time needed in the growth of bacterial culture is the time required for a cell to divide or population to double (Benjamin C., 2004). Most bacteria have a doubling time of 1-3 hours, although some may be greater than 24 hours. E. coli may have a doubling time of 20 minutes; get 20 generations in 7 hours, going from one cell to one million cells. As the cell grows, they will follow the curve of growth which will distinct into four phases’ accordance to the region in the graph shown in Figure 1. First region is lag phase; the first major phase of microbial growth in a batch fermentation process. Here, a period of adaptation of the cells to their new environment and minimal increase in cell density. May be absent in some fermentations. Second region called exponential phase; the second major phase of microbial growth in a batch fermentation process which is also known as the logarithmic growth phase. Cells here have adjusted to their new environment and are divided at a constant rate resulting in an exponential increase in the number of cells present. This is known as the specific growth rate and is represented mathematically by first order kinetics as the following: dX =( μ−k d ) X … … … … … … …..(1) dt where X is the cell concentration, μ is the cell growth rate, and k d is the cell death rate. The term (μ- kd) can be referred to as μnet. The cell death rate is sometimes neglected if it is considerably smaller that the cell growth rate. Cell growth rate is often substrate limited, as depicted in the figure to the right. The growth curve is well represented by Monod batch kinetics, which is mathematically depicted in following equation: μ=
μ max S … … … … … … … … …(2) Ks+S
where μ is the specific growth rate μmax is the maximum specific growth rate, S is the growth limiting substrate concentration, and Ks is the saturation constant which is equal to the substrate concentration that produces a specific growth rate equal to half the maximum specific growth rate. All specific growth rates account for the term (μ- k d) and should be considerate to be μnet value.
Figure 4.1: Phases of microbial growth (Benjamin C. 2004)
As stated by Shuler & Kargi (2002), there are other models used to determine cell growth raet that depend upon inhibition such as substrate inhibition, product inhibition, and toxic compounds inhibition. The type of inhibition causes mathematical changes in the previously presented Monod equation for batch kinetics. Substrate inhibition; that can occur during the initial growth phases while substrate concentrations are high. If this is a major problem, continuous or fed-batch fermentation methods should be considered. Then, the product inhibition can occur after induction of the recombinant gene. Stationary phase which is the third phase of microbial growth in a batch fermentation process happen when the number of cells dividing and dying is in equilibrium and can be the result of the following; depletion of one or more essential
growth nutrients, accumulation of toxic growth associated by-products, and also stress associated with the induction of recombinant gene. Stationary phase has a primary metabolite, or growth associated, production stops and also secondary metabolite, or growth associated, production may continue. The death phase; the fourth major phase of microbial growth in a batch fermentation process also known as the decline phase. The rate of cells dying is greater than the rate of cells dividing as similar to exponential phase; it is represented mathematically by first order kinetics as the following: dX =−k d X … … … … … … … … .(3) dt 5.0
APPARATUS 1. Microbe: Escherichia coli 2. Shake flask (250mL flasks and 1000mL flasks) 3. Eppendorf tubes/ falcon tube (1.5mL) 4. Cuvettes (spectrophotometer) 5. Thermostat rotary shaker/ incubator shaker 6. Refrigerated centrifuged 7. Media (for specific microbe) 8. Ethanol (70% ethanol for swabbing for sterility) 9. Spectrophotometer 10. Bunsen burner for sterility 11. Graduated flask for measuring media (1000mL, 100mL, 10mL) 12. Laminar flow hood for sterility 13. Biochemical analyser 14. HPLC for product measurement like ethanol 15. Cotton plugged 16. pH meter
6.0
PROCEDURE
(i) Preparation of media 1. Microorganism used was Escherichia coli (E-coli). Terrific Broth was chosen as a readied phosphate buffer media. 2. 47.60 g of Terrific Broth was weighed and need to be filled up to 1L volume Glycerol as carbon source (4mL/1L)
3. The media was autoclaved at 121oC for 20 minutes. Glycerol and media were autoclaved together. (ii) Preparation of cell culture a) Seed culture preparation (inoculum) 1. 5 loops of grown E-coli were taken on agar plates and added to the sterilized media of 150mL in 1000mL shake flask. 2. The media was grown at 350 rpm for 5 hours at 37oC, assuming exponential growth of E-coli. OD for seed culture was recorded by using spectrophotometer. b) Main experiment 1. 10% of inoculum was transferred by using aseptic technique to the main experiment media. 2. The shake flask was capped (cotton plugged) and swabbed with 70% ethanol before incubation in a thermostated rotary shaker at required rotational speed and temperature for 24 hours. (iii) Sampling 1. Required amount of sample was transferred into the sampling tube with interval time for every hour or every 4 hours. 2. 5mL of sample was withdrawn every time sampling was done during fermentation for measuring optical density (OD) and total cell number (biomass concentration: g/L). a) Absorbance Analysis (Optical Density) (OD) 1. 1 mL of sample was transferred into a cuvette and the optical density measurement was made using a spectrophotometer with the wavelength set at 600nm. 2. The spectrophotometer was calibrated to zero by blank consisting 1 mL Terrific Broth. 3. With 1 mL sample, using micropipette, 100 uL sample being added to 900 uL of Distilled Water for OD measurement in 1000 uL Cuvette. b) Cell Dry Weight. (Biomass Concentration) (X) (g/L)
1. Dried centrifuge tubes were weighed and initial mass was recorded. (empty 2. 3. 4. 5.
container) 1 mL sample was added to weighted centrifuge tube. The sample was centrifuged at 10,000 rpm and at T of 25oC for 20 minutes The supernatant was taken out. The centrifuge tube was dried (left with biomass only) in oven at 80 oC for
overnight 6. The dried centrifuged tubes were left in desiccator. 7. The centrifuge tube was weighed and the final mass was recorded (with biomass = Cell Dry Weight) Cell Dry Weight = Final mass – Initial mass
7.0
RESULTS
Seed Inoculum No 1
Time (h) 0
OD (10 times dilution) 0.268
Real OD 1.344
Main Experiment Table 7.1: Experimental data for Terrific broth
No
Time (h)
Absorbance Optical Density, OD
Absorbance Optical Density,
Empty Centrifuge
Dried Centrifuge tube +
Cell Dry Weight X
1 2 3 4 5 6 7 8 9 10 11 12 13
(100x dilution) ODread 0.174 0.564 0.769 1.426 1.569 1.659 1.695 2.000 1.987 2.214 2.271 2.247 2.246
0 1 2 3 4 6 8 10 12 14 16 18 20
Real OD (ODread times 100) 1.626 1.661 1.777 2.197 2.355 2.442 2.569 2.616 2.687 2.801 2.904 2.914 2.936
(m1)
sample (m2)
(g/L) (m2 – m1)
1.0701 1.0921 1.0837 1.0800 1.0946 1.1027 1.0933 1.0917 1.0964 1.0903 1.0989 1.0996 0.9813
1.0751 1.0996 1.0918 1.0860 1.1027 1.1090 1.1000 1.0976 1.1035 1.0988 1.1096 1.1094 0.9925
0.0050 0.0075 0.0081 0.0060 0.0081 0.0063 0.0067 0.0059 0.0071 0.0085 0.0107 0.0098 0.0112
Absorbance value for Optical Density 25 20 15
Time (h)
f(x) = 8.62x - 5.04 R² = 0.79
10 5 0 0
0.5
1
1.5
2
Absorbance, OD
Figure 7.1: Graph of Absorbance optical density, OD versus time.
2.5
Absorbance Real OD 3.5 3
f(x) = 0.06x + 1.85 R² = 0.85
2.5 2
Time (h)
1.5 1 0.5 0 0
5
10
15
20
25
20
25
Absorbance
Figure 7.2:Graph of absorbance real OD versus time.
Cell Dry Weight Curve 0.01 0.01 f(x) = 0x + 0.01 R² = 0.49
0.01
Time (h)
0.01 0 0 0 0
5
10
15
Cell Dry Weight (g/L)
Figure 7.3: Graph of cell dry weight versus time.
8.0
SAMPLE OF CALCULATION
Cell dry weight: Cell Dry Weight=( Dried centrifuge tube+ sample )−( Empty centrifuge) ¿ ( 1.0976−1.0917 ) g ¿ 0.0059 g
9.0
DISCUSSION
The main objective of this experiment is to investigate the kinetic growth of microorganism. The cell used to be experimented is E-coli and is cultivated inside a shake flask. This process can be called simple fermentation. The media supplies nutrients which contain carbon sources for the microorganism. The flask is shaken during cultivation process in order for the cell and media to mix to increase its homogeneity and providing aeration. The culture is gone through 24 hours of cultivation. Within 24 hours, the biomass/cell sample is taken out for every 3 hours to analyse the concentration of the cell (g/L), the cell dry weight and the glucose concentration. Furthermore, in order to get the concentration of the cell inside the flask, a sample is taken and is placed in a cuvette. The cuvette containing sample is used in the spectrophotometer to obtain its absorbance density of the optical reading. It is proved that the higher the absorbance reading, the higher number of cell presence during that particular time. Based on the results obtained, it can be seen that the reading of the
absorbance increases with time until at hour of 12 th, it decreases a bit and start increases again at hour of 14th. For the real absorbance, it increases as time increases until 24 hours is finished. It indicates that the number of cell increase throughout the cultivation showing that the cell is growing. In contrast, the decrease in cell number in 12 th hour shows that the cell growth has reached its deceleration phase where the growth of the cell is started to slow down. The decelerating growth phase is where the culture is in a transient state. During this stage there are feed/back mechanisms that regulate the bacterial enzymes involved in key metabolic steps to enable the bacteria to withstand starvation. There is much turnover of protein for the culture to cope with this period of low substrate availability. In cell growth, the cell will go through several phases like lag, exponential, deceleration, stationary and death phase. Besides that, another analysis that can be performed to analyse the cell sample is by taking the dry weight of the cell. Before starting the experiment, the falcon tube needs to be weight. During this time, a sample is taken out of the cultivation flask and transferred to a falcon tube. The tube is being centrifuged to separate the supernatant with the cell. The separated cell is being dried in the oven overnight. After that, the tube is weighed to determine its dry cell weight at every interval hour sample is being taken. Based on the results, it can be seen that the dry cell weight shows a pattern of increasing and decreasing of dry weight cell throughout the 24 hours of experiment. Based on the facts, the cell dry weight should increase when the number of cell increased inside the shake flask. But in this experiment, the results obtained do not show this pattern. This may happen due to the fact that the empty falcon tube weighed is not accurate which will affect the cell dry weight.
10.0
CONCLUSION
At the end of the experiment we can ensure that, E-coli is suitable to be fermented inside a shake flask as it is a simple method used to investigate the growth kinetics of the microorganism. It is essential to determine when to harvest the culture for different purposes. Several analyses on the cell need to be done to know the growth kinetics of the cell and the duration for each phase as the microorganism will go through several phases
in their growth. It includes the cell concentration, glucose concentration and also the cell dry weight analyses. This method can be done in the laboratory before the fermentation or the cultivation of microbes in large scale is done. During the course growth, the cells is continuously changing and adapting itself in the media environment. It also continuously changes in physical and chemical conditions. As the conclusion, the microbial culture in batch culture system that is in shake flask system depends on the end product desired. The substrate concentration in the culture medium and growth parameters changes correspondingly throughout the growth phases. Although we fail to achieve the objectives of the experiment due to some errors during conducting the experiment, we try to learn from our mistakes for the next experiment.
11.0
RECOMMENDATION
1. The supernatant of cell concentration should be taken out carefully without any taking out of the biomass. 2. Aseptic technique must be practised when handling biomass concentration to avoid any contamination. 3. Cuvette must be wiped cleanly to prevent any scratch that would affect the spectrophotometer reading during protein test. 4. The cap of the viral must be opened to fasten the drying process of the biomass in the oven. 5. Disinfect the work area with 70% alcohol before handling the culture. 6. This experiment must be carried out under the laminar flow to prevent any contamination to the culture.
12.0
REFERENCES/APPENDICES
1. Benjamin C. (2004). Chapter 6: Microbial Growth, Pearson Education Inc. Retrieved November 11, 2016 from http://classes.midlandstech.edu/carterp/courses/bio225/chap06/Microbial%20Growth %20ss5.html 2. Briggs, G. E., and Haldane, J. B. (2000). A Note on the Kinetics of Enzyme Action, Biochem J 19. 3. Friedrich Widdel (June 05, 2010). Journal of Theory and Measurement of Bacterial Growth.
Retrieved
by
November
10,
2016
from
http://www.mpi-
bremen.de/Binaries/Binary13037/Wachstumsversuch 4. Michael L. Shuler & Fikret Kargi (2002). Introduction to Fermentation. Bioprocess Engineering: Basic Component, 2nd ed. 161-178. Prentice Hall PTR. 5. Stephanie C., Stephanie J. et al. (2014). Chapter 6: Fermentation and Enzyme Technologies in Food Processing. Food Processing: Principle and Application, 2nd ed. 107-137. John Wiley & Sons, Ltd.
